Project Summary/Abstract Understanding the role of the tumor suppressor gene PTEN in human disease is challenging because its protein product controls many aspects of cellular physiology, including cell division, DNA repair, and cytoskeletal organization. This challenge is exemplified by the myriad of phenotypes observed in patients with germline PTEN variants and the difficulty in classifying clinically-sequenced PTEN missense variants as pathogenic versus benign. In light of these difficulties, this project aims to systematically catalogue the effect of thousands of PTEN missense variants on multiple cellular phenotypes, with the goals of better understanding the role of PTEN in disease and helping classify clinically-observed PTEN missense variants. To do this, three high-throughput functional assays ? testing for cell growth, resistance to genotoxic agents, and cellular morphology ? will be employed in cultured human cell lines. By examining the relationships between these cancer-related cellular phenotypes, PTEN missense variation, and clinical outcomes, it will be possible, for the first time, to comprehensively dissect the role of PTEN in germline inherited disease and somatic cancers. First, a high-throughput, growth-based functional assay will be used to measure the effect of a library of nearly all possible PTEN missense variants for their ability to inhibit cellular growth, the primary PTEN function thought to be important for human disease. The growth assay will be repeated in the presence of genotoxic agents to measure the effect of PTEN variants in protecting against DNA damage. Second, a microscope-based selection for cell morphology will be performed using a new technology we developed that analyzes millions of cells and separates them based on microscopic visual phenotypes. Each of these high-throughput assays will annotate each missense variant with a quantitative score documenting the effect of that variant on the selected phenotype. Third, once the data are collected, the relative contribution of each PTEN function to disease will be examined by comparing which functions are lost in variants annotated as pathogenic in ClinVar and in variants commonly found in tumors. These high-throughput experiments will be completed under the supervision of Dr. Douglas Fowler, who pioneered large-scale, sequencing-based functional assays. The lab has already published a paper quantifying the effect of PTEN missense variants on a much simpler phenotype, protein abundance, and so already has the tools and knowledge required for studying this critical tumor suppressor. Courses that focus on the responsible conduct of research and the biometric statistical analyses will be pursued to ensure the reproducibility and proper analysis of the data. Additional help with interpreting the findings? clinical utility will be provided by Dr. Mary- Claire King, who works with patients harboring PTEN variants, and by Dr. Colin Pritchard, who is the head of Lab Medicine at the University of Washington and oversees the clinical sequencing core.